Abstract

Gemcitabine (2′, 2′-difluoro-2′-deoxycytidine; dFdCyd) has been shown to
be a potent radiosensitizer in tumor cells both in vitro
and in vivo. We evaluated the ability of dFdCyd to
enhance the radiosensitivity of two human glioblastoma cell lines. The
results demonstrated that U251 cells were more sensitive to the
cytotoxicity of dFdCyd, and that dFdCyd was able to radiosensitize
these cells. In contrast, D54 cells were more resistant to the
cytotoxic effect of dFdCyd, and no radiosensitization occurred at any concentration of dFdCyd tested. Because radiosensitization by
dFdCyd has been correlated with its ability to deplete dATP pools
through inhibition of ribonucleotide reductase by dFdCyd diphosphate,
we evaluated the metabolism of dFdCyd in both cell lines. At equitoxic
concentrations of dFdCyd, both cell lines accumulated similar levels of
the cytotoxic metabolite, dFdCyd triphosphate, as well as similar
levels of dFdCyd monophosphate in DNA. In U251 cells,
radiosensitizing concentrations of dFdCyd (10 or 25 nm;
IC10 or IC50) depleted dATP by ∼80% within
4 h. In contrast, 80 nm (IC50) was unable
to deplete dATP by >30% within 4 h in D54 cells. Higher
concentrations of dFdCyd or hydroxyurea, an inhibitor of ribonucleotide
reductase that depleted dATP >90%, also did not produce
radiosensitization in D54 cells. D54 cells were not resistant to
radiosensitization because bromodeoxyuridine was able to induce
radiosensitization. Because D54 cells express wild-type p53, whereas
U251 cells express a mutant p53, the effect of dFdCyd and ionizing
radiation on cell cycle progression was evaluated. Radiation alone
produced a G1 block in D54 cells and a transient
G2-M block in U251 cells. After a 24 h incubation with
dFdCyd alone or in combination with ionizing radiation, U251 cells
readily accumulated in S-phase, which remained elevated for at least
72 h, consistent with previous results in other mutant p53 cell
lines. In addition, radiation enhanced the ability of dFdCyd to induce
S-phase-specific cell death in U251 cells. In contrast, D54 cells
showed a G1 block after dFdCyd and radiation exposure, with
fewer cells in S-phase for at least 48 h after drug
washout/irradiation. Furthermore, treatment with dFdCyd and/or
radiation did not increase the amount of S-phase-specific cell death in
D54 cells compared with control cells. These results suggest that the
G1 block in D54 cells resulting from wild-type p53
induction prevented radiosensitization by dFdCyd.

INTRODUCTION

dFdCyd
3
(gemcitabine) is a deoxycytidine analogue that has shown clinical
activity in the treatment of solid tumors, including pancreatic and
non-small cell lung cancer
(1,
2,
3,
4,
5)
. As a nucleoside
analogue, after transport into the cell
(6)
, dFdCyd
requires phosphorylation for its antitumor activity, with the initial
phosphorylation by deoxycytidine kinase being the rate-limiting step in
its activation
(7, 8)
. Mechanistic studies have
demonstrated that there are two major pathways through which dFdCyd is
toxic to tumor cells: (a) formation of the triphosphate,
dFdCTP, can inhibit DNA synthesis directly or interfere with
replication through incorporation of dFdCMP into the elongating DNA
strand; and (b) the diphosphate, dFdCDP, is a potent,
mechanism-based inhibitor of ribonucleotide reductase that results in
the depletion of the necessary deoxynucleoside triphosphates for DNA
synthesis
(9,
10,
11,
12,
13,
14)
. dFdCyd can potentiate its own
cytotoxicity through several mechanisms. For example, the
dFdCyd-mediated depletion of dCTP can enhance the incorporation of
dFdCMP into DNA, and the ability of dFdCTP to inhibit dCMP deaminase
can prevent its catabolism
(13, 15, 16)
. The cytotoxicity
of dFdCyd in different cell lines is dependent upon the extent to which
each of these pathways is affected by dFdCyd nucleotides
(17)
.

On the basis of the observation that other antimetabolites that inhibit
ribonucleotide reductase can enhance radiation-induced
cytotoxicity
(18,
19,
20)
, we hypothesized that dFdCyd
would function as a radiation sensitizer. Indeed, it has now been
demonstrated that dFdCyd is among the most potent of
radiosensitizers in vitro(21,
22,
23)
. Recent
studies in vivo have confirmed these observations and have
shown significant tumor growth delay with the combination of dFdCyd
and ionizing radiation in animal models
(24,
25,
26)
. These
results have prompted a variety of clinical trials using dFdCyd as a
radiosensitizer for tumors in which the lack of local control is the
reason for clinical failure, such as head and neck cancer, pancreatic
cancer, non-small cell lung cancer, and gastrointestinal malignancies
(27,
28,
29)
.

Glioblastoma multiforme is an aggressive brain tumor with poor
prognosis in patients because of its propensity to recur locally.
Despite a multitude of efforts to impact the natural course of this
disease with surgery, chemotherapy, and/or radiation therapy, the
median survival for these patients is <1 year
(30)
. One
strategy to improve therapeutic outcome has been to use
radiosensitizers
(31, 32)
. Of these, the most promising
agents were the halogenated thymidine analogues; however, this
combination has proven toxic to patients
(33)
. dFdCyd may
be preferable to the halogenated thymidine analogues as a
radiosensitizer based on its potent activity at low doses in
vitro. In addition, dFdCyd can radiosensitize cells in
vitro, even after a brief exposure
(34)
, compared
with the lengthy dosing required for the thymidine analogues
(35)
, suggesting that the once-weekly dosing schedule of
dFdCyd in patients will be sufficient for radiosensitization.

We have evaluated the ability of dFdCyd to enhance radiation-induced
cytotoxicity in the U251 and D54 human glioblastoma cell lines.
Although both cell lines were sensitive to the cytotoxic effects of
dFdCyd, only the U251 cells were radiosensitized by the drug. We noted
that the U251 cells expressed a mutant p53, as did the other cell types
reported previously by us to be radiosensitized by dFdCyd (HT-29 human
colon carcinoma, and the BxPC-3 and Panc-1 pancreatic cancer cell
lines), whereas D54 cells express wild-type p53
(36,
37,
38)
.
Mutations in p53 or allelic loss of chromosome 17p occur commonly in
the development of human glioblastomas
(39)
. Inactivating
p53 mutations have been shown to occur in >40% of adult glioblastomas
(40, 41)
. Because induction of wild-type p53 by
DNA-damaging agents can lead to cell cycle arrest
(42)
,
and cell cycle position affects cytotoxicity induced by radiation, we
considered the possibility that the p53 status of cells may affect
their ability to be radiosensitized by dFdCyd. These two cell lines
provided an opportunity to evaluate the metabolism of dFdCyd and its
effects on cell cycle progression to gain a greater understanding of
the factors necessary to produce radiosensitization with this
nucleoside analogue. A preliminary description of these results was
reported previously
(43)
.

MATERIALS AND METHODS

Chemicals.

dFdCyd and dFdCTP were synthesized and generously provided by Eli Lilly
and Co. (Indianapolis, IN). RNase A was purchased from Boehringer
Mannheim (Indianapolis, IN). All other chemicals were of the highest
purity available.

Cytotoxicity Assays.

Cytotoxicity was measured using a standard colony formation assay. Cell
culture flasks (25 cm2) were plated with between
300,000 and 600,000 cells a minimum of 36 h prior to the addition
of drug. Exponentially growing cells were incubated with drug for 4 or
24 h. At the conclusion of the drug incubation period, cells were
washed with Dulbecco’s PBS, trypsinized, and counted using a Coulter
(Hialeah, FL) electronic particle counter. Approximately 100 viable
cells were plated into each 35-mm diameter well of a six-well culture
dish and allowed to grow in the absence of drug for 10–14 days. At
that time, the resulting colonies were fixed using a methanol:glacial
acetic acid solution (3:1, v/v) and stained with 0.4% crystal violet.
Colonies of >30 cells were counted, and survival was determined as a
fraction of plating efficiency of untreated control cells. The control
plating efficiency for both cell lines was ∼40%.

Radiosensitization Assays.

After drug and/or radiation treatment, cells were assessed for
clonogenic survival as described above. Radiation survival data from
drug-treated cells were corrected for plating efficiency by comparison
to cells treated with drug alone. Cell survival curves were fit using
the linear quadratic equation. Radiation sensitivity is expressed in
terms of the mean inactivation dose, which represents the area under
the cell survival curve
(44)
. Radiosensitization is
expressed as the RER, which is defined by the mean inactivation dose
(radiation treatment)/mean inactivation dose (drug + radiation treatment).

Irradiation of Cells.

Monolayer cultures of either U251 or D54 cells were irradiated at 1–2
Gy/min using 60Co (AECL Theratron 80). Dosimetry
was performed using an ionization chamber connected to an electrometer
system that was directly traceable to a National Institute of Standards
and Technology standard. All cells were irradiated at room temperature.

Analysis of dNTP Pools.

Cells were incubated with drug for 1–24 h, harvested by
trypsinization, and counted. The nucleotides were extracted with
ice-cold 0.4 n perchloric acid and neutralized with 10
n KOH. The majority of the ribonucleotides were removed
from the deoxyribonucleotides by elution over a boronate affinity
column as described previously
(45)
. Deoxyribonucleotides
were separated and quantitated by strong anion exchange HPLC using a
Waters (Milford, MA) gradient system composed of two model 501 pumps, a
U6K injector, and a model 996 photodiode array detector. This system
was controlled by Millennium 2010 software. Before injection, each
sample was centrifuged at 14,000 × g for 2
min and acidified to pH 2.8. Samples were then injected onto a 5-μm
Partisphere 4.6 × 250-mm SAX column (Whatman
Scientific, Hillsboro, OR) and eluted with a linear gradient of
ammonium phosphate buffer ranging from 0.15 m (pH
2.8) to 0.6 m (pH 2.8–3.8) at a flow rate of 2
ml/min. Nucleotides were identified and quantitated by comparison to a
known amount of authentic standards using their characteristic
absorbance spectra over the range of 200–350 nm.

Cell Cycle Analysis.

Flow cytometric analysis was performed as described in Hoy et
al.(46)
. Briefly, at the conclusion of the
dFdCyd incubation, cells were pulse labeled with 30μ
m BrdUrd for 15 min and then harvested by
trypsinization, counted, and washed with PBS. Cells were then fixed in
cold 70% ethanol at a concentration of 1,000,000 cells/ml, with
samples not to exceed a total of 3,000,000 cells. Fixed cells were
stored at 4°C for up to 10 days. Within 6 h prior to flow
cytometric analysis, fixed cells were washed with PBS and resuspended
in 1 ml of PBS containing 0.5 mg/ml RNase A and incubated for 30 min at
37°C. Cells were then washed with PBS, resuspended in 1 ml 0.1
n HCl containing 0.7% Triton X-100, and
incubated for 10 min on ice. This was followed by another PBS wash,
resuspension in 1 ml of sterile HPLC grade water, and incubation at
95°C for 15 min. The samples were immediately transferred to an
ice-water bath for an additional 15 min. Cells were then washed with
PBS containing 0.5% Tween 20. One hundred μl of PBS containing 0.5%
Tween 20 and 5% calf serum (PBT) were added to each cell pellet,
followed by the addition of 100 μl of anti-BrdUrd mouse
IgG1 antibody (1:100 dilution; PharMingen, San Diego, CA)
and incubation for 30 min at room temperature. After centrifugation,
150 μl of FITC-conjugated, goat antimouse IgG antibody (1:20–35
dilution; Sigma Chemical Co, St. Louis, MO) were added to the pellet,
mixed gently, and incubated for 30 min at room temperature. Samples
were centrifuged and resuspended in 0.5 ml of 18 μg/ml PI containing
40 μg/ml RNase A. Trout erythrocyte nuclei (Biosure, Grass Valley,
CA) were added as an internal standard. Treated cells were placed in
the dark a minimum of 30 min prior to cell cycle analysis using a
Coulter EPICS Elite ESP flow cytometer. Cell cycle data were further
analyzed using WinMDI software (version 2.8.8) provided by Joseph
Trotter of The Scripps Research Institute.

Apoptosis.

Apoptosis was determined by sub-G1 content, as
indicated by flow cytometry. Briefly, adherent cells were harvested by
trypsinization, counted, and washed with PBS. Cells were then fixed in
cold 70% ethanol at a concentration of 1,000,000 cells/ml with samples
not to exceed 3,000,000 cells. Cells were fixed a minimum of 1 h
prior to the addition of 0.5 ml of 18 μg/ml PI containing 40 μg/ml
RNase A. Trout erythrocyte nuclei were added as an internal standard.

RESULTS

Cytotoxicity of dFdCyd.

The sensitivity of U251 and D54 cells to dFdCyd alone was determined to
select the appropriate concentration range in which to evaluate dFdCyd
as a radiosensitizer in these two cell lines. Using a clonogenic
survival assay, U251 cells (IC50, 21.4 ± 1.1 nm) were found to be at least 3-fold more
sensitive than D54 cells (IC50, 78.3 ± 17 nm) after a 24 h incubation with dFdCyd. At
higher concentrations, U251 cell survival was 1–2 logs less than that
of D54 cells (Fig. 1)
⇓
.

Cytotoxicity of dFdCyd in human glioblastoma cells. U251
(▪) or D54 (•) cells were incubated with dFdCyd for 24 h and
assayed for clonogenic survival. Values shown represent the mean of
triplicate determinations calculated from a single experiment;
bars, SE. Experiments were repeated at least three
times.

Radiosensitization by dFdCyd.

Previous work from this lab has shown that dFdCyd is a potent
radiosensitizer in solid tumor cells, such as human colon carcinoma and
pancreatic cancer cells
(22, 23)
. To determine the ability
of dFdCyd to enhance the sensitivity of these cells to radiation,
noncytotoxic (IC10) and cytotoxic
(IC50) doses of dFdCyd for 24 h were
evaluated. Survival at the IC10 (90.9 ± 9.3% survival) was not found to be significantly different
from untreated cells (100.0 ± 3.2% survival), and it
permitted standardization of the noncytotoxic dose. Consistent with
previous studies in other solid tumor cell lines, U251 cells were
radiosensitized after a 24 h exposure to either 10 nm
(IC10) or 25 nm
(IC50) dFdCyd. The RERs were 1.60 ± 0.03 and 1.77 ± 0.22, respectively (Fig. 2
⇓
and Table 1
⇓
).

Effect of dFdCyd on the sensitivity of human glioblastoma
cells to ionizing radiation. A, U251 cells were treated
with either no drug (control, •) or 10 nm dFdCyd
(IC10, □) for 24 h, followed by the indicated doses
of radiation. B, D54 cells were treated with either no
drug (control, •) or 80 nm dFdCyd (IC50, □)
for 24 h, followed by radiation. Cell survival was determined by
colony formation assay. The surviving fraction was corrected for cell
death attributable to drug alone. Data shown are from a representative
experiment. Each experiment was repeated at least three times.

U251 or D54 cells were treated with the indicated doses of
radiosensitizing agents for 24 h. Values represent a
mean ± SE for at least three experiments.

In contrast to the U251 cells, D54 cells were not radiosensitized using
either noncytotoxic or cytotoxic doses of dFdCyd for 24 h (RERs<1.0; Fig. 2
⇓
and Table 1
⇓
). Higher concentrations of dFdCyd incubated
for only 4 h in D54 cells also failed to produce
radiosensitization (Fig. 3)
⇓
. The lack of radiosensitization of D54 cells is not due to an apparent
radioresistance. D54 cells are significantly more sensitive to the
effects of ionizing radiation alone than U251 cells, as indicated by D
bar values of 2.01 ± 0.06 and 2.61 ± 0.08 for D54 and U251 cells, respectively (P < 0.001; Ref.
44
).

Comparison of the radiation enhancement ratio in human
glioblastoma cells. Either U251 (▪) or D54 (□) cells were treated
with dFdCyd, as indicated, followed by radiation. Values are means of
at least three determinations; bars, SE. ∗,
P < 0.02 (value is significantly>
1.0).

Metabolism of dFdCyd.

To determine whether an altered rate of metabolism of dFdCyd could
account for the lack of radiosensitization in D54 cells, dFdCTP levels
were measured in the two cell lines. Both cell lines were incubated
with their respective 24 h IC50 of dFdCyd,
and the dFdCTP pool was measured periodically. Under these conditions,
dFdCTP accumulation was similar, with peak levels of 0.08 and 0.06
nmol/107 cells at 12 h for U251 and D54
cells, respectively (data not shown). In addition, dFdCMP incorporation
into DNA was similar in both cell lines after a 24 h incubation
with IC50 concentrations of dFdCyd
(0.40 ± 0.04 versus 0.57 ± 0.07 pmol/107 cells in U251 and D54 cells,
respectively).

Effect of dFdCyd on dATP.

Previous studies in human colon carcinoma and pancreatic cancer cell
lines suggested that radiosensitization by dFdCyd was related to its
ability to deplete the endogenous dATP in the cells by at least 90%
because of inhibition of ribonucleotide reductase
(22, 23)
. It was important to determine whether the lack of
radiosensitization in D54 cells was attributable to an inability to
deplete dATP. Both cell lines were treated for 24 h with the
IC50 of dFdCyd, and the nucleotide pools were
measured. Within 4 h, dATP was depleted to <0.04
nmol/107 cells, or to 20% of control levels in
U251 cells. The amount of dATP continued to decrease, with 0.01
nmol/107 cells (4%) remaining at 12 h and
0.001 nmol/107 cells (0.5%) at 24 h. In D54
cells, dFdCyd depleted dATP by only 30% within 4 h and required
24 h to deplete dATP to a minimum level of 0.07
nmol/107 cells (12%; Fig. 4
⇓
). In addition, dGTP was depleted to a greater extent in U251 cells than
D54 cells under these conditions. However, by the end of the 24 h
incubation, the dGTP level in U251 cells began to recover (data not
shown). No significant differences in the other dNTP pools were
observed after dFdCyd treatment in these two cell lines.

Effect of dFdCyd on the dATP pool in human glioblastoma
cells. Exponentially growing U251 (▪) or D54 (•) cells were treated
with 25 nm dFdCyd (IC50) or 80 nm
dFdCyd (IC50), respectively, for 24 h. Cellular
nucleotides were extracted with perchloric acid, eluted over a boronate
affinity column to remove ribonucleotides, and analyzed by HPLC. Data
are expressed as the means from at least two separate experiments;
bars, SE.

Because dFdCyd at the IC50 concentration was
unable to deplete dATP to <10% of control levels in D54 cells, we
examined the ability of hydroxyurea, another known radiosensitizer that
also inhibits ribonucleotide reductase, to deplete dATP. Treatment of
D54 cells with 0.6 mm hydroxyurea (24 h
IC50) did not produce radiosensitization (RER,
1.06 ± 0.04), although it did deplete dATP to <2%
(<0.01 nmol/107 cells) of control levels within
2 h of drug addition (data not shown). In contrast, 3
mm hydroxyurea (24 h IC50) was
able to radiosensitize U251 cells (RER, 1.39 ± 0.08).
Hydroxyurea depleted dATP in U251 cells in a similar pattern and to a
similar extent as dFdCyd (data not shown).

To determine whether there was a difference in dATP levels in these two
cell lines after the combined treatment of dFdCyd and ionizing
radiation, both cell lines were treated for 4 h with higher
concentrations of dFdCyd (4 h IC50) and then
irradiated with 5 Gy. Immediately after irradiation, both cell lines
were markedly depleted of dATP, but neither showed recovery of dATP or
changes in the other dNTP pools with the next 4 h, the time period
during which the majority of DNA double strand breaks induced by
radiation are repaired
(34)
.

Effects of BrdUrd.

Because D54 cells could not be radiosensitized by two agents that
inhibit ribonucleotide reductase, it was important to determine whether
D54 cells could be radiosensitized by agents that radiosensitize via
alternate mechanisms. BrdUrd was chosen because it has been shown that
incorporation of BrdUMP, 5′-monophosphate of BrdUrd, into DNA increases
sensitivity to radiation damage
(47)
. BrdUrd was evaluated
as a radiosensitizer at both noncytotoxic and cytotoxic concentrations
for 24 h. U251 cells were radiosensitized at a noncytotoxic dose
(IC10; RER, 1.71 ± 0.19; Table 1
⇓
). BrdUrd was also able to radiosensitize D54 cells at a noncytotoxic
dose (IC10; RER, 1.19 ± 0.06),
and a cytotoxic dose (IC50; RER, 1.84 ± 0.16). Thus, D54 cells were not resistant to all
radiosensitizers.

Expression of p53 and mdm-2.

One notable difference between these cell lines is that U251 cells
express a mutant p53, whereas D54 cells express wild-type p53
(36,
37,
38)
. Because induction of wild-type p53 in response
to DNA damage can alter the cell cycle distribution, we evaluated the
effect of dFdCyd and radiation on p53 expression. U251 or D54 cells
were treated with their IC50 concentrations of
dFdCyd for 24 h, followed by 5 Gy of ionizing radiation, and
Western blot analysis was performed using an antibody that can detect
both mutant and wild-type p53. These studies verified that p53 was
constitutively expressed at a relatively high level in the U251 cells,
as expected for a mutant p53 cell line. In contrast, p53 was present at
lower levels in untreated D54 cells as compared with untreated U251
cells (data not shown). Treatment of D54 cells with dFdCyd alone did
not induce p53 expression during the 24 h exposure prior to
irradiation (data not shown). However, after the subsequent
irradiation, p53 was induced within 1 h (Fig. 5)
⇓
, indicative of functional p53. Irradiation without prior dFdCyd
treatment also induced expression of p53.

Western blot analysis for p53 (top) and
mdm-2 (bottom). Either U251 (left) or D54
(right) cells were incubated with dFdCyd
(IC50) for 24 h and then irradiated with 5 Gy of
ionizing radiation. T = 0 represents the
conclusion of the 24 h drug incubation when dFdCyd was washed out
and the cells were irradiated.

mdm-2 is a negative regulator of p53 that blocks transcriptional
activation and mediates degradation by binding to p53 and promoting
ubiquitinization
(48)
. mdm-2 is expressed at higher levels
in human glioblastoma cell lines
(49)
. The response of
mdm-2 to the combined treatment of dFdCyd and radiation was measured by
Western blot analysis. mdm-2 was not detected in U251 cells after drug
and radiation treatment, as predicted for a mutant p53 cell line. In
D54 cells, mdm-2 was expressed within 4 h of radiation treatment,
after an increase in p53 protein (Fig. 5)
⇓
. At the time of mdm-2
induction, there also was a modest reduction in p53 protein levels,
consistent with a functional feedback mechanism. mdm-2 levels remained
elevated for 20 h after the initial induction after irradiation.

Cell Cycle Progression.

Because p53 is an important cell cycle checkpoint regulator, it was
essential to determine whether this difference in p53 status altered
the ability of cells to progress through the cell cycle after treatment
with dFdCyd and radiation. U251 cells were treated with 25
nm dFdCyd for 24 h and/or 5 Gy of radiation. The
medium was replaced immediately after radiation treatment, and the cell
cycle was monitored by dual parameter flow cytometry, which measured
both BrdUrd incorporation and DNA content, for the following 72 h.
In response to 5 Gy (IC90), U251 cells show a
typical G2-M block within 12 h, and
G2-M remained elevated at 24 h (Fig. 6
⇓
and Table 2
⇓
). This G2-M block was partially released by
48 h, as indicated by a decreased percentage of cells in
G2-M, an increase in G1 and
in S-phase, and an increased cell number. At 72 h,
G2-M remained elevated above control levels, and
the total cell number increased further. In contrast, after a 24 h
treatment with dFdCyd alone, U251 cells accumulated in S-phase
(>70%), with corresponding decreases in G1 and
G2-M. After drug washout, U251 cells slowly began
to progress through the cell cycle. The S-phase population decreased by
24 h after drug washout, and there was a large increase in
G2-M after 48 h. The increase in
SNI [non-BrdUrd incorporating cells with S-phase
DNA content identified as dying/dead cells, similar to the findings of
Pallavicini et al.(50)]
to ∼12% at
24 h after drug washout indicated that S-phase-specific death was
induced by dFdCyd in agreement with the observed loss in cell number
(data not shown). U251 cells treated with 25 nm
dFdCyd for 24 h, followed by 5 Gy, show almost the same cell cycle
pattern as with dFdCyd alone; however, SNI
increased to ∼33% at 24 h after drug washout and was
consistently higher at 48 and 72 h compared with cells treated
with dFdCyd alone.

Effects of dFdCyd and ionizing radiation on cell cycle
distribution of U251 (A) and D54 (B)
cells. U251 cells were treated with 25 nm dFdCyd and/or 5
Gy of ionizing radiation. D54 cells were treated with 80 nm
dFdCyd and/or 5 Gy of ionizing radiation. Control samples were obtained
from untreated cells. Drug was added 24 h prior to irradiation.
Cells were irradiated and/or drug was removed at
T = 0 h. Thirty μm
BrdUrd was added during the last 15 min prior to the indicated time
points. Cells were prepared for dual parameter flow cytometry to
determine DNA content and BrdUrd as described in “Materials and
Methods.” Results of a single experiment are displayed. Experiments
were repeated at least three times.

Effect of dFdCyd and ionizing radiation on the cell cycle distribution
of human glioblastoma cells

U251 and D54 cells were treated as indicated. Drug was added 24 h
prior to T = 0 h (−24 h).
T = 0 represents the time of irradiation
and/or drug washout. DNA and BrdUrd content were measured using dual
parameter flow cytometry. SNI represents cells that
have S-phase DNA content by PI staining but do not incorporate BrdUrd
(a measurement of S-phase-specific cell death). Data are from a
representative experiment repeated at least three times.

D54 cells treated with 5 Gy (IC95) displayed an
increase in G2-M to ∼18% within 12 h
after irradiation (Table 2)
⇓
. However, by 24 h, the majority of
this population had progressed into G1. The
increase in G1 and decrease in S-phase indicated
that the cells were blocked in G1. D54 cells
treated with 80 nm dFdCyd (IC50) for
24 h exhibited a cell cycle distribution similar to controls
throughout the 72 h following drug washout. In response to the
combination of dFdCyd and radiation, G1 increased
to ∼74% within 24 h of irradiation/drug washout and remained
elevated for the following 48 h, similar to treatment with
radiation alone. As observed with radiation alone, the percentage of
cells in S-phase decreased. The low S-phase percentage was associated
with no change in cell number. Furthermore, the addition of radiation
to dFdCyd treatment did not increase SNI. These
results show that the majority of D54 cells were unable to enter into
S-phase for at least 48 h after the combination treatment of
dFdCyd and radiation.

Apoptosis.

Expression of wild-type p53 can lead to apoptotic cell death in
response to dFdCyd, radiation, and other DNA-damaging agents
(51, 52)
. Therefore, we evaluated the ability of dFdCyd and ionizing
radiation to induce apoptosis in U251 and D54 cells at 0, 24, 48, and
72 h after drug/radiation treatment. In response to dFdCyd alone,
U251 cells readily undergo apoptosis, as measured by
sub-G1 DNA content determined with flow
cytometry. Treatment with 10 nm (24 h
IC10), 25 nm (24 h
IC50), or 120 nm (24 h
IC99) dFdCyd led to 16.9–21.1% of the cell
population undergoing apoptosis 24 h after drug washout (data not
shown). Seventy-two h after washout, the percentage of apoptotic cells
was dependent on the severity of treatment (Fig. 7)
⇓
. U251 cells treated: with 10 nm dFdCyd showed a decline in
apoptosis by 72 h; with 25 nm dFdCyd plateaued at 28%
apoptotic cells; and with 120 nm dFdCyd displayed apoptosis
in up to 46.5% of the population. U251 cells displayed lower levels of
apoptosis with cytotoxic treatments of ionizing radiation. Neither 5 Gy
(IC90) nor 10 Gy (IC99.5)
produced >22.3% apoptosis during the 72 h after irradiation.
Combinations of dFdCyd and radiation that produced radiosensitization
in U251 cells produced apoptosis in up to 27.9% of the cell
population; however, there was not even an additive increase compared
with the individual treatments.

Apoptosis in human glioblastoma cells in response to
dFdCyd and ionizing radiation. Either U251 (
) or D54 (▪) were
treated as indicated, and apoptosis was measured as sub-G1
content by flow cytometry 72 h after irradiation and drug washout.
Values are the means of at least three determinations;
bars, SE. †, not done.

In contrast to the U251 cells, D54 cells do not readily undergo
apoptosis in response to treatment with dFdCyd and/or radiation.
Incubation with 80 nm dFdCyd produced similar levels of
apoptosis, as seen in untreated cells, up to 72 h after drug
removal. Increasing the dose of dFdCyd to 760 nm (24 h
IC99) induced apoptosis to a greater extent
(22.7%), but this required up to 72 h after the washout of
dFdCyd. D54 cells showed an even less tendency to die through apoptosis
after treatment with radiation than did U251 cells. Although these
cells are more sensitive to radiation treatment, there was no
appreciable increase (<5%) in apoptotic cell death in response to
either 5 Gy (IC95) or 10 Gy
(IC99.5) as compared with controls. Finally,
combining dFdCyd with radiation in D54 cells still did not produce an
increase in apoptotic cell death as compared with the individual
treatments at any of the time points examined (Fig. 7)
⇓
.

DISCUSSION

dFdCyd is a promising radiosensitizing agent for the treatment of
patients with solid tumors. In vitro studies have
demonstrated that dFdCyd is a potent radiosensitizer, and in
vivo studies in animals have shown that the combination of dFdCyd
and ionizing radiation produces significant tumor growth delay
(24,
25,
26)
. On the basis of these results, clinical trials
have begun with the combination of dFdCyd and ionizing radiation
(27,
28,
29)
. However, there has not been an extensive
analysis of the mechanism by which dFdCyd is able to enhance
radiation-induced cytotoxicity. Earlier work from this laboratory
demonstrated a correlative relationship between the duration of dATP
depletion in the cell and the extent of radiosensitization
(22)
. Here we have extended these studies using two human
glioblastoma cell lines, of which only one was radiosensitized by
dFdCyd. The results suggest that, in addition to dATP depletion, the
ability of cells to progress into S-phase after dFdCyd and radiation
treatment may be key for radiosensitization to occur.

Both the U251 and D54 cell lines were sensitive to the cytotoxic effect
of dFdCyd at low nanomolar concentrations. However, U251 cells were at
least 3-fold more sensitive than the D54 cells. Analysis of the
phosphorylation of dFdCyd indicated that similar levels of dFdCTP
accumulated at equitoxic concentrations in the two cell lines,
suggesting that the difference in cytotoxicity may be explained by
altered rates of dFdCyd metabolism. Alternatively, noting the
difference in p53 status and ability to undergo apoptosis between the
two cell lines, it may be interesting to explore whether the lack of
expression of wild-type p53 in the U251 cells sensitizes them to dFdCyd
and facilitates p53-independent apoptosis. A recent report using
glioblastoma cell lines indicated that sensitivity to dFdCyd
cytotoxicity did not differ between cells expressing either mutant or
wild-type p53
(53)
. However, this study used cell lines
that did not originate from a single parental line or compared
sensitivity after forced expression of wild-type p53 in a cell line
with a mutant p53 background. It may be of interest to compare dFdCyd
sensitivity in matched cell lines with wild-type or mutant p53
expression.

Evaluation of cytotoxicity from the combination of dFdCyd and ionizing
radiation demonstrated that U251 cells were radiosensitized at both the
IC10 and IC50
concentrations of dFdCyd. However, attempts to radiosensitize D54 cells
failed using a variety of dFdCyd doses and incubation periods.
Initially, we hypothesized that this lack of radiosensitization was due
to the inability of dFdCyd to deplete the dATP pool in D54 cells. Upon
initial inspection, this appeared to be true, because there was a>
80% reduction in dATP in U251 cells compared with <30% reduction
in D54 cells within 4 h using equitoxic doses of dFdCyd (Fig. 4)
⇓
.
This suggested that the remaining level of dATP in D54 cells after
dFdCyd treatment was sufficient to prevent radiosensitization. However,
further depletion of dATP to <10% of the control level using
hydroxyurea failed to produce radiosensitization in D54 cells, although
hydroxyurea was able to effect a similar decrease in dATP and
radiosensitize U251 cells. Therefore, in D54 cells, dATP depletion
alone was not sufficient to promote radiosensitization by dFdCyd or
hydroxyurea. The D54 cells were not resistant to radiosensitization by
all agents, because BrdUrd resulted in significant radiosensitization.
Furthermore, incorporation of dFdCMP into DNA cannot explain the lack
of radiosensitization because D54 cells were able to incorporate
slightly more dFdCMP than U251 cells at equitoxic doses of dFdCyd.

Although a strong association has been made between dATP depletion and
radiosensitization for dFdCyd in numerous cell lines
(22, 23, 54)
, the mechanism by which low dATP levels may effect
radiosensitization is not clear because radiosensitizing concentrations
of dFdCyd in other cell lines did not produce DNA double strand breaks
or inhibit their repair
(34, 55)
. Low dATP levels may
result in errors of replication, such as insertion of an incorrect
nucleotide for the missing dATP, and the D54 cells may be able to
prevent or correct this error. Thus, this cell line may be important in
determining the molecular target for radiosensitization with dFdCyd.

An alternative explanation for the difference in radiosensitization
with dFdCyd is that the two cell lines respond to DNA damage in
different ways. A variety of DNA-damaging agents can induce expression
of wild-type p53, resulting in increased transcription of proteins such
as p21, mdm-2, bax, and GADD45 which, in turn, can determine whether a
cell will continue to progress through the cell cycle, arrest in
G1 or G2, repair DNA
damage, or die via apoptosis
(42)
. As predicted for a
wild-type p53 cell line, D54 cells exhibited a competent
G1-S cell cycle checkpoint in response to dFdCyd
and ionizing radiation. This may allow D54 cells time to repair DNA
damage and/or prevent the replication of damaged cells after
irradiation. In contrast, the mutant p53-expressing U251 cells
continued to progress into S-phase and G2-M after
dFdCyd treatment and irradiation. This cell cycle pattern is similar to
that reported previously for HT-29 human colon carcinoma cells, a cell
line that also expresses a mutant p53 protein, after a 2 h
treatment with a noncytotoxic dose of dFdCyd
(34)
. Reports
in the literature suggest that the p53 status of a cell can affect its
inherent radiosensitivity
(56,
57,
58,
59,
60,
61)
. Here we have observed
a difference in cell cycle progression between the radiosensitive U251
cells and the nonradiosensitized D54 cells, which may be attributable
to their difference in p53. The ability of the U251 cells to continue
to progress through the cell cycle after damage from dFdCyd and
radiation may lead to the synergistic enhancement of cell death that
presents as radiosensitization.

The observed difference in cell cycle progression resulted in a
significant difference in cell cycle distribution of the two cell lines
at the time of irradiation. Greater than 70% of the U251 cells were in
S-phase, whereas <37% of the D54 cells were in S-phase. These results
are similar to those reported previously by us using the HT-29 human
colon carcinoma cell line
(22, 34)
. In addition, a recent
report using synchronized cell populations indicated that cells must be
in S-phase to be radiosensitized by dFdCyd
(62)
. Because
cells in S-phase are generally more resistant to ionizing radiation
than cells at the G1-S border or in
G2-M, radiosensitization of U251 cells by dFdCyd
is not attributable to the redistribution into a more
radiosensitive phase
(18)
. However, this
redistribution may be important for radiation to enhance the
S-phase-specific cell death induced by dFdCyd, as was observed
prominently in the U251 cells but was noticeably absent in D54 cells.
This enhancement of S-phase cell death may be responsible for
radiosensitization by dFdCyd.

In the studies presented here, it is interesting to note that p53 was
readily induced by ionizing radiation, yet a 24 h exposure to the
IC50 of dFdCyd did not induce p53 in D54 cells.
This is consistent with the lack of effect on the cell cycle
distribution of D54 cells with drug treatment alone. A recent report
indicated that dFdCyd alone induced wild-type p53 expression in H460
human lung cancer cell lines, although this was measured after a
72 h incubation with IC50 and
IC80 concentrations of drug
(63)
. It
is possible that the ability to induce p53 by dFdCyd varies by cell
line.

Several reports have demonstrated that dFdCyd can induce apoptosis in a
variety of cell types
(53, 63, 64)
. In addition, compared
with lower grade malignant brain tumors, glioblastomas in patients are
associated with higher levels of apoptotic cells (reviewed in Ref.
65
). Therefore, it was important to analyze the effect of
dFdCyd and ionizing radiation on the ability of these cells to undergo
apoptosis. Although a high percentage of U251 cells became apoptotic
after treatment with dFdCyd alone, ionizing radiation was less able to
induce apoptosis, and the combination of these agents did not increase
apoptosis as compared with the single-agent treatments. These results
are consistent with previous reports from other laboratories,
indicating that radiation is not a strong inducer of apoptosis in
nonlymphoid cell lines (reviewed in Ref.
66
). D54 cells
were less likely to undergo apoptosis after either dFdCyd or ionizing
radiation than U251 cells, and the combination did not result in an
increase in the amount of apoptotic cells in the D54 cell line. Thus,
in both cell lines, the addition of radiation did not increase the
ability of dFdCyd to produce apoptosis. Although these two cell lines
differed in their ability to undergo apoptosis with dFdCyd and/or
ionizing radiation, radiosensitization was associated with a decreased
incidence of apoptosis in the U251 cells. Therefore, the lack of
radiosensitization in D54 cells does not appear to be related to low
induction of apoptosis.

The data presented here support the previous findings that, in addition
to dATP depletion, progression of cells into S-phase after dFdCyd
treatment is important for radiosensitization. When cells accumulated
in S-phase, radiation enhanced the S-phase-specific cell death induced
by dFdCyd. With the prominent G1 block observed
after the combination of dFdCyd and ionizing radiation in the D54 cells
and the difference in cell cycle distribution at the time of
irradiation in these two cell lines, it is tempting to speculate that
expression of wild-type p53 prevents radiosensitization through
inhibition of progression into S-phase. However, in consideration of
the myriad cellular effects triggered by p53, it is possible that the
effects on cell cycle progression are secondary to a primary effect of
p53 on another requisite but undefined pathway for dFdCyd-mediated
radiosensitization. If expression of p53 is involved in the lack of
radiosensitization in the D54 cells, then its inactivation should allow
the cells to be radiosensitized. This could be accomplished by
introducing the human papillomavirus E6 protein to promote degradation
of p53 or by using a p53 antisense construct. This hypothesis is
currently under investigation.

Acknowledgments

We gratefully acknowledge Mark A. KuKuruga of the University of
Michigan Flow Cytometry Core facility for excellent technical advice.

Footnotes

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

↵1 This work was supported in part by Grant CA
83081 from the NIH, University of Michigan-Comprehensive Cancer Center
NIH Grant CA46592, University of Michigan-Multipurpose Arthritic Center
NIH Grant AR20557, and the University of Michigan Core Flow
Cytometry facility.